Abstract The establishment and maintenance of epithelial tight junction integrity is essential to the normal function of epithelial organs;above all, the ability of the intestinal epithelium to serve as a selective barrier to antigens and pathogens while absorbing nutrients is fundamental to intestinal function. Also, tight junctions together with associated polarity complexes are critical for the maintenance of epithelial polarity. Our investigations of epithelial development and polarity have focused on the endosomal protein, endotubin. Endotubin is an integral membrane protein that is resident in apical endosomes of polarized epithelial cells. It is expressed at high levels in developing intestine, particularly when the enterocytes are establishing polarity. Moreover, we have evidence that endotubin regulates junctional integrity and epithelial polarity through interaction with aPKC and Rab14. In this proposal, we will elucidate the mechanism of action of endotubin and Rab14 in the establishment and maintenance of epithelial tight junctions. Experiments outlined in this proposal will define the motifs of endotubin critical for the establishment and maintenance of epithelial junctions and for interaction with binding partners. Our hypothesis is that endotubin serves as a scaffolding protein to organize and target junctional and polarity proteins from the apical endosomes. Furthermore, we hypothesize that loss of endotubin function could result in loss of barrier function and/or apical-basolateral polarity, leading to compromised immunity in the newborn, increased susceptibility to inflammatory bowel disease, and/or cancer.